Primer system and applications thereof
A technology of nucleotide sequences and expression vectors, applied in the primer system and its application fields, can solve the problems of unfavorable environment, high cost, waste of living materials, etc.
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[0033] This example is used to illustrate the acquisition of CFB
[0034] 1. Overlap PCR to obtain the target gene
[0035] Synthesize the following four primers:
[0036] P1: Add a restriction endonuclease KpnI enzyme cutting site (GGTACC) and a protection base CGG to the 5' end of primer 1 shown in SEQ ID No: 1;
[0037] P2; as shown in SEQ ID No: 2;
[0038] P3; as shown in SEQ ID No: 3;
[0039] P4; at the 5' end of primer 4 shown in SEQ ID No: 4, a restriction endonuclease XhoI restriction site (CTCGAG) and a protective base CGG are added;
[0040] PCR reaction system: (Pfμmixtμre was purchased from Bao Biological Engineering (Dalian) Co., Ltd.)
[0041]
[0042]
[0043] PCR reaction program: pre-denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 45s, annealing at 60°C for 30s, extension at 72°C for 30s, and 35 cycles; after the cycle, extend at 72°C for 10 minutes and cool to 4°C.
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